Anti‐Helicobacter pylori activity of Fagopyrum Tataricum (L.) Gaertn. Bran flavonoids extract and its effect on Helicobacter pylori‐induced inflammatory response

Abstract Tartary buckwheat flavonoids have a variety of effects on anti‐inflammatory, anti‐oxidation, as well as anti‐tumor and are valuable for academic research and industrial application. Helicobacter pylori (H. pylori) infection is associated with various gastrointestinal diseases in humans, and an increase in its resistance has led to the failure of many drugs. In this study, we quantified the main monomers of tartary buckwheat (Fagopyrum Tataricum (L.) Gaertn.) bran flavonoids extract through HPLC analysis. Then, we investigated the anti‐H. pylori activity and the effect on cell inflammation of tartary buckwheat flavonoids extract and its four main flavonoid monomers (rutin, quercetin, kaempferol, and nicotiflorin). The results showed that tartary buckwheat flavonoids extract and its four flavonoid monomers could inhibit the growth of H. pylori and down‐regulate the expression of proinflammatory factors IL‐6, IL‐8, and CXCL‐1 in H. pylori‐induced GES‐1 cells. Moreover, we also confirmed that tartary buckwheat flavonoids extract could reduce the expression of virulence factor gene of H. pylori. In summary, tartary buckwheat can alleviate the cell inflammation induced by H. pylori, which provides a theoretical basis for the development of tartary buckwheat healthcare products.


| INTRODUC TI ON
Helicobacter pylori (H. pylori) is gram-negative bacteria that planted in the stomach and duodenum of humans and may cause gastritis, peptic ulcers, and gastric cancer (Kim et al., 2021). It is prevalent in about 50% of the global population and is a major carcinogen of gastric cancer (Huang et al., 2021). Proinflammatory cytokines such as IL-6, IL-8, and CXCL-1 in gastric epithelial cells and immune cells can be promoted by H. pylori infection (Wu et al., 2017). Various virulence factors produced by H. pylori induce the production of these cytokines (Alzahrani et al., 2014). Reducing or eradicating the inflammatory reaction will help promote ulcer healing and reduce the risk of gastric cancer. Today, first-line standard triple therapy of two antibiotics and a proton-pump inhibitor is the most widely used treatment to eradicate H. pylori infection. However, this approach is costly, has significant side effects (such as nausea, vomiting, and diarrhea), and increases H. pylori resistance because of the overuse of antibiotics (Boyanova et al., 2016;Fallone et al., 2016;González et al., 2021).
Therefore, seeking alternative therapies to reduce H. pylori infection and treat gastrointestinal diseases is the direction that researchers continue to explore.
As early as 1982, doctors had been using natural products based on empirical knowledge to combat these diseases (Yu et al., 2022). Research suggests that over 240 plant species have shown anti-H. pylori activity (Salehi et al., 2018). Tartary buckwheat (Fagopyrum Tataricum (L.) Gaertn.) is a food crop with rich nutritional value and widely distributed throughout southwest China (Tomotake et al., 2006). Its planting area and production in China is the highest in the world, and it has been popularized in many countries around the world (Holasova et al., 2002). As raw material for food, Fagopyrum Tataricum has been processed into buckwheat tea, flour, and noodles (Zhang et al., 2012). There are many bioactive components in tartary buckwheat, among which flavonoids are the most important ones. Flavonoids, rich in fruits, vegetables, tea, and medicinal plants, are highly effective antioxidants and have received great attention and extensive research (Rodríguez De Luna et al., 2020). Flavonoids also have the functions of preventing cardiovascular and cerebrovascular diseases, resisting cancer, antiaging, analgesic, and hemostasis (Chen & Zhang, 2021). In addition, studies have reported that some flavonoids (especially chalcones) show up to sixfold stronger antibacterial activities than standard drugs in the market (Farhadi et al., 2019). Kaempferol exerts a positive influence on the inflammatory response caused by H. pylori infection and could down-regulate proinflammatory cytokines (Yeon et al., 2019). However, no studies have focused on the antibacterial activity and anti-inflammatory properties of Fagopyrum Tataricum bran extract against H. pylori.
In this article, we prepared Fagopyrum Tataricum bran flavonoids extract under optimal extraction conditions and quantified the main flavonoid monomers in it. Then, we investigated their antibacterial activity and anti-inflammatory properties against H. pylori in human gastric epithelial cells (GES-1 cells).

| Extraction and identification of flavonoids
In preliminary experiments, we determined the optimal macroporous resin ADS-7 and the optimal separation and purification process of tartary buckwheat bran flavonoids (the sample flow rate was 2 BV/h, the concentration of the sample solution was 1.0 mg/ mL, the washing volume was 2 BV, the eluent concentration of food grade ethanol was 90%, the elution flow rate was 2 BV/h, and the elution volume was 6 BV). We validated the process by magnifying it 24 times (stage two) and 240 times (stage three), respectively.
Besides, we detected standard solutions of the four flavonoid monomers by the UV-Visible Spectrum ranging from 200 nm to 500 nm, respectively, and selected the appropriate wavelength according to the maximum absorption peak. A high-performance liquid chromatography (HPLC) system (LC-16, Shimadzu) was used to quantitative determination of main components in tartary buckwheat bran flavonoids extract.

| H. pylori strains and culture conditions
The two strains of H. pylori (SS1 and 26695) were derived from Key Laboratory of Biological Resource and Ecological Environment of Chinese Education Ministry. Four clinical H. pylori strains (SCU-HP-0916A, SCU-HP-0916C, SCU-HP-1230A, and SCU-HP-1230B) were from Sichuan Provincial People's Hospital. All H. pylori strains were cultured on an anaerobic incubator (5% O 2 , 10% CO 2 , and 85% N 2 ) at 37°C for 3 days with a Columbia blood agar plate containing brain heart infusion broth, 7% defibrinated sheep blood, and 0.1% antibiotics . in paper disks were 200 mg/mL and 50 mg/mL, respectively. After a 3-day cultivation, the H. pylori of the blood agar plate was eluted into 1 mL sterile saline. Bacterium solution was diluted to a turbidity of 1 MacFarlane (∼3 × 10 8 CFU/mL) by nephelometer (WGZ-2XJ).

| Determination of inhibition zones
Next, a 90 μL of the diluted suspension was swabbed evenly over the surface of a new blood agar plate with a sterile cotton swab. After slightly drying, put the paper disks to the surface of the plates and then culture at 37°C for 3 days. The inhibitory zone diameter was measured by criss-cross method and each set of experiments was performed in triplicate.

| Determination of minimum inhibitory concentration (MIC)
The MIC of tartary buckwheat flavonoids extract against H. pylori strains was determined by the agar dilution method. Flavonoids extracts from tartary buckwheat were added to a 24-well plate containing 2 mL Columbia medium at the concentrations of 200, 100, 50, 25, 12.5, and 6.25 mg/mL, respectively. Blank control group and negative control group were blood plates without tartary buckwheat flavonoids extract. After the plates were solidified, 100 μL of bacterial suspension (1 × 10 8 CFU/mL) was added to each well of the experimental group and the negative control group. Finally, the plates were incubated at 37°C in an anaerobic incubator for 3 days, and the lowest concentration at which showed H. pylori growth was considered as MIC. Each set of experiments was performed in triplicate.

| Cell culture
The source of human gastric epithelial cells (GES-1 cells) and their culture conditions refer to the article by Li et al. (2020).

| Treatment of GES-1 cells with H. pylori, tartary buckwheat flavonoids extract, and flavonoid monomers
3 × 10 5 cells were seeded in 12-well plates in each well. The experiment was divided into six groups in triplicate: blank control group, and H. pylori co-treatment group: 500 μg/mL tartary buckwheat flavonoids extract and 100 μg/mL four flavonoid monomers were added in GES-1 cells suspension; at the same time, H. pylori was infected with the MOI of 100:1. All groups were cultured at 37°C with 5% CO 2 .

| Quantitative real-time PCR (qRT-PCR)
Total RNAs of H. pylori and cells were extracted with Trizol reagent. RNA concentration was determined using BioDrop, and then cDNA was obtained by reverse transcription using PrimeScript RT reagent Kit (RR047A, TaKaRa). qRT-PCR was performed using an iQ5 Real-Time PCR detection system (Quant Studio 3, Thermo). The amplification was programed to start at 95°C for 30 s, 40 cycles of degeneration at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 20 s. The primers were designed using Primer 5.0 software and are listed in Table 1.

| Statistical analysis
All analysis results were described as mean ± standard deviation (SD). The data were analyzed with GraphPad Prism 8.0 (GraphPad TA B L E 1 qRT-PCR primer sequences.

Name
Sequence (

| Identification of main flavonoid monomers in tartary buckwheat bran extract
As can be seen from Figure

| UreA and UreB gene expression decreased in H. pylori after adding tartary buckwheat flavonoids extract
As shown in Figure

| DISCUSS ION
The rate of H. pylori infection is increasing, affecting more than half the people in developed countries and 80 percent in underdeveloped countries (Azami et al., 2017). In order to overcome the problem of drug resistance, we urgently need to find alternative drugs to treat urease is an important pathogenic factor of H. pylori (Guo et al., 2014).
Actually, the activity of urease helps H. pylori survive in an acidic environment. It has been shown that inhibition of urease can prevent H. pylori from adhering to gastric mucosa, which is very important for the treatment of H. pylori-related diseases. Besides, inhibiting the activity of urease can inhibit the growth of H. pylori. Lee et al. (2018) have

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.